Use of G-CSF to reduce acute rejection

ABSTRACT

Reduction of occurrence of acute rejection of organ transplants is achieved by treatment with a G-CSF protein product.

BACKGROUND OF THE INVENTION

Organ transplants of liver, kidney, heart, and lung are now regularlyperformed now as treatment for end-stage organ disease. Allograft (samespecies donor and recipient) as well as xenograft (different speciesdonor and recipient) transplants have been performed. Two primaryproblems for all organ transplants, however, have been acute rejectionof the donor organ and the high risk of infection. Treatment for acuterejection, intensification of immunosuppression, causes deterioration ofimmune function, resulting in increased susceptibility to seriousinfection.

Organ transplants evoke a variety of immune responses in the recipient.In acute rejection, the graft is initially invaded by recipientmononuclear cells (macrophage, lymphocyte and monocyte cells). If thesecells perceive antigenic differences in the graft, they will process andpresent the antigen to a T-lymphocyte and activate it in anantigen-specific manner. The T-cell then stimulates the central lymphoidsystem to elicit an immune response. The response is usually acombination of cellular (T-cell mediated) and humoral (B-cell mediated)responses. The former reaction appears to be the primary cause of theinitial acute transplant rejection occurring one to three weekspost-transplant. The outcome of this acute rejection depends in part onwhether immunosuppressive treatment is effective.

Acute rejection is reported to occur in 50 to 70% of hepatic grafts,depending on the criteria for diagnosis. Although quite common, fewtransplanted livers fail because of uncontrollable acute rejection.Relative ease of acute rejection control of hepatic grafts is not seenwith other solid organ grafts, such as kidney, pancreas, and cardiacgrafts.

In liver transplant patients, acute rejection is characterized by twoconsecutive days of rising bilirubin or liver enzymes (such as SGOT,SGPT, and alkaline phosphatase), which would indicate graft dysfunction,and simultaneous histologic findings of rejection on biopsy. Theearliest histologic changes characteristic of acute rejection areaccumulation of mononuclear cells in the portal tracts. The infiltrateconsists of lymphocytes, and to a lesser extent neutrophils andeosinophils. Infiltrates that spill over into the parenchyma constitutea more specific sign of established acute rejection. The presence ofeosinophils and the polymorphonuclear cells (PMNs) is often obscured bya prominent lymphocytic infiltrate. Eosinophilia and endothelialitis ofthe central vein and portal vein are also seen. Histologic evidence ofbiliary damage is reported to occur in 10 to 75% of patients with acuterejection. See Foster et al., Transplantation, 47:72-74 (1989) andWilliams et al., Seminars in Liver Disease, 12:60-72 (1992).

In heart transplant patients, acute rejection is characterized byclinical signs of fever, arrhythmia, congestive heart failure, andincreased cardiac volumes on echocardiogram. The diagnosis isestablished by transvenous endomyocardial biopsy using grading criteriapublished by Billingham, J. Heart Transplant, 9:272-276 (1990).

In lung transplant patients, acute rejection is characterized byclinical signs of fever, leukocytosis, bronchorrhea, and increasingalveolar to arteriolar oxygen gradient, all in the absence of pulmonaryinfection. Radiographic findings on chest X-ray may be normal or mayshow bi-perihilar infiltrates. Spirometry typically shows decreasedforced expiratory volume over one second. The final diagnosis isestablished on clinical grounds, by response to bolus steroids, and onthe basis of transbronchial biopsy. These criteria have been discussedin Paradis et al., J. Heart and Lung Transplant, 11:S232-6 (1992).

In kidney transplant patients, acute rejection is characterized bydeteriorating renal function as shown by increasing BUN and creatinine,graft enlargement, fever, oliguria, hypertension, and reduced renalclearances. Renal scans will initially show a reduction in excretionwith cortical retention, followed in several days by reductions incortical uptake as well. If the rejection episode occurs during a periodof acute tubular necrosis, its diagnosis may be delayed, being madeeither by serial scan assessment or by a transplant biopsy during afebrile episode. Lymphocymria is often found and may be helpful, alongwith a negative urine culture in ruling out graft pyelonephritis. Renalbiopsies performed at this time typically reveal interstitial nephritis,mononuclear cell infiltrate, acute arteritis, and glomerular injury.Patients with multiple or severe early rejections have worse graftfunctional outcomes (at one, two, and five years) than patients without.

Granulocyte colony stimulating factor (G-CSF) is one of thehematopoietic growth factors, also called colony stimulating factors,that stimulate committed progenitor cells to proliferate and to formcolonies of differentiating blood cells, G-CSF preferentially stimulatesthe growth and development of neutrophils, and is useful for treating inneutropenic states. Welte et al., PNAS-USA 82: 1526-1530 (1985); Souzaet al., Science 232: 61-65 (1986) and Gabrilove, J. Seminars inHematology 26: (2) 1-14 (1989). G-CSF increases the number ofcirculating granulocytes and has been reported to ameliorate infectionin sepsis models. G-CSF administration also inhibits the release oftumor necrosis factor (TNF), a cytokine important to tissue injuryduring sepsis and rejection. See, e.g., Wendel et al., J. Immunol.,149:918-924 (1992).

In humans, endogenous G-CSF is detectable in blood plasma. Jones et al.,Bailliere's Clinical Hematology 2 (1): 83-111 (1989). G-CSF is producedby fibroblasts, macrophages, T cells trophoblasts, endothelial cells andepithelial cells and is the expression product of a single copy genecomprised of four exons and five introns located on chromosomeseventeen. Transcription of this locus produces a mRNA species which isdifferentially processed, resulting in two forms of G-CSF mRNA, oneversion coding for a protein of 177 amino acids, the other coding for aprotein of 174 amino acids. Nagata et al., EMBO J 5: 575-581 (1986). Theform comprised of 174 amino acids has been found to have the greatestspecific in vivo biological activity. G-CSF is species cross-reactive,such that when human G-CSF is administered to another mammal such as amouse, canine or monkey, sustained neutrophil leukocytosis is elicited.Moore et al. PNAS-USA 84: 7134-7138 (1987).

Human G-CSF can be obtained and purified from a number of sources.Natural human G-CSF (nhG-CSF) can be isolated from the supernatants ofcultured human rumor cell lines. The development of recombinant DNAtechnology has enabled the production of commercial scale quantities ofG-CSF in glycosylated form as a product of eukaryotic host cellexpression, and of G-CSF in non-glycosylated form as a product ofprokaryotic host cell expression. See, e.g., U.S. Pat. No. 4,810,643(Souza) incorporated herein by reference.

G-CSF has been found to be useful in the treatment of indications wherean increase in neutrophils will provide benefits. For example, forcancer patients, G-CSF is beneficial as a means of selectivelystimulating neutrophil production to compensate for hematopoieticdeficits resulting from chemotherapy or radiation therapy. Otherindications include treatment of various infectious diseases and relatedconditions, such as sepsis, which is typically caused by a metabolite ofbacteria. G-CSF is also useful alone, or in combination with othercompounds, such as other cytokines, for growth or expansion of cells inculture for example, for bone marrow transplants. G-CSF has beenadministered to transplant patients as an adjunct to treatment ofinfection or for treatment of neutropenia. See Diflo et al., Hepatology,16:PA278 (1992), Wright et al., Hepatology, 14:PA48 (1991), Lachaux ellal., J. Pediatrics, 123:1005-1008 (1993), and Colquhoun et al.,Transplantation, 56:755-758 (1993).

SUMMARY OF INVENTION

The present invention provides a method for reducing the occurrence ofacute rejection of organ transplants in a patient comprisingadministering a therapeutically effective dose of G-CSF protein product.In preferred forms of practice of the invention, recombinant human G-CSFis administered in unit dosage forms of from 5 to 50 μg/kg on a dailybasis.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, administration of G-CSF proteinproduct to subjects after organ transplantation results in a reducedrate of acute rejection of the organ transplant, as well as reducedinfection rates and improved overall survival rates. Treatment accordingto the invention also results in reduced duration of stay underintensive care, reduced duration of ventilator treatment, and reducedincidence of acute respiratory distress syndrome (ARDS). G-CSF proteinproduct can be administered intravenously according to the inventiongenerally at dosages varying between 5 and 50 μg/kg/day over a period of10 to 14 days. G-CSF protein product can also be administered via oral,pulmonary or other routes.

The term "G-CSF protein product" as used herein is defined as naturallyoccurring human and heterologous species G-CSF, recombinantly producedG-CSF that is the expression product consisting of either 174 or 177amino acids, or fragments, analogs, variants, or derivatives thereof asreported, for example in Kuga et al., Biochem. Biophy. Res. Comm 159:103-111 (1989); Lu et al., Arch. Biochem. Biophys. 268: 81-92 (1989);U.S. Pat. Nos. 4,810,643, 4,904,584, 5,214,132, and 5,218,092; EP 0335423; EP 0 272703; EP O 459630; EP O 256843; EP O 243153; WO 9102874;Australian Application document Nos. AU-A-10948/92 and AU-A-76380/91.Included are chemically modified G-CSFs, see, e.g., those reported in WO9012874, EP 0 401384 and EP 0 335423. See also, WO 9315211; WO 9305169;JP 04164098; WO 9206116; WO 9204455; EP O 473268; EP O 456200; WO9111520; WO 9105798; WO 9006952; WO 8910932; WO 8905824; WO 9118911; andEP O 370205.

The invention is better understood by reference to the followingillustrative examples wherein: Example 1 demonstrates that, in humanliver transplant patients, augmenting the usual treatment protocol withadministration of a G-CSF protein product results in a statisticallysignificant reduction in acute rejection rates and infection rates, incomparison to patients not treated with G-CSF; and Example 2demonstrates that, in a rat heterotopic heart transplant model of acuterejection, administration of a G-CSF protein product to the rats,without other treatment, resulted in a statistically significantimprovement in graft survival.

EXAMPLE 1

Recombinant human G-CSF Filgrastim (NEUPOGEN®, Amgen Inc., ThousandOaks, Calif.) was administered to high-risk, adult liver transplantpatients to study its effects on infection and rejection. Thirty-fourconsecutive liver allograft recipients were treated with G-CSF inaddition to the usual protocol immunosuppressive therapy and wereprospectively monitored over at least 120 days for sepsis and rejectionoutcomes. The data were compared to the previous forty-nine consecutiveliver transplant patients who had not received G-CSF. The two groupswere similar for age, sex, cause of liver failure, pre-opChilds-Turcotte grading, pre-op infection incidence and creatinine,United Network for Organ Sharing (UNOS) status, length of surgicalprocedure and blood components transfused.

All patients were treated pre-operatively with the antibioticsvancomycin and Claforan. Post-operatively, the antibiotic treatment wascontinued for 72 hours and the usual immunosuppressive regime wasinstituted. The usual immunosuppression protocol consists of 2.5 mg/kgCyclosporine intravenously every twelve hours, regulated to maintain ablood level of cyclosporine at 200 to 400 ng/ml, and 1.5 mg/kgSolu-Medrol intravenously daily, with the dose decreasing by 10% everyday until reaching a level of 0.35 mg/kg. At 7 to 10 dayspost-transplant, after the T-tube was clamped, the patients wereswitched to oral cyclosporine at a total dose of 10 to 15 mg/kg/day,divided in two doses and titrated to reach the same therapeutic bloodlevel, and oral prednisone at 0.35 mg/kg/day.

Standard protocol diagnosis and treatment of infection and rejection wasemployed. Infection was diagnosed by (1) a temperature of greater than101.5° F. or less than 96° F., (2) tachycardia, and (3) a specific siteof infection as shown by positive sputum, blood, urine or woundcultures, and was treated with appropriate antibiotics. Acute rejectionwas diagnosed by serological and histological methods as described aboveand was treated with 1 g Solu-Medrol once a day for two consecutivedays.

The G-CSF-treated patients also received human recombinant G-CSFintravenously at a dose of 5-10 μg/kg/day for 10 days postoperatively,with the dosage titrated to maintain a blood absolute granulocyte count(AGC) between 10,000 and 20,000 cells/mm³. The outcome results aredisplayed in Table I below.

                  TABLE I                                                         ______________________________________                                                    Controls  G-CSF                                                   Results     (N = 49)  (N-34)    Significance                                  ______________________________________                                        AGC pre-op  4.4 ± 2.3                                                                            4.5 ± 2.3                                                                            NS t-test                                     (× 10.sup.3)                                                            AGC peak post-op                                                                          9.4 ± 4.5                                                                            20.5 ± 6.5                                                                           p < 0.0001 t-test                             ICU stay (days)                                                                           20.5 ± 26.1                                                                           8.0 ± 11.8                                                                          p < 0.002 t-test                              Vent.time (days)                                                                          17.3 ± 25.6                                                                          5.1 ± 8.2                                                                            p < 0.004 t-test                              ARDS        37%       11%       p < 0.004 x.sup.2                             Acute rejection                                                                           51%       30%       p < 0.02 x.sup.2                              Chronic rejection                                                                         10%        9%       NS x.sup.2                                    Infections/patient                                                                        2.3 ± 2.8                                                                            1.5 ± 1.3                                                                            p < 0.006 t-test                              Survival (120 days)                                                                       76%       91%       p < 0.06 Breslow                              ______________________________________                                    

The G-CSF-treated patients had significantly reduced rates of infection(1.5 on average compared to 2.3 without G-CSF treatment) and acuterejection (30% compared to 51%, which was statistically significantusing chi-square analysis).

EXAMPLE 2

The effect of G-CSF (Neupogen) treatment was investigated in theheterotopic rat heart transplant model described in Ono et al., J.Thoracic Cardiovasc. Surg., 57:225 (1969). 24 Lewis rats underwentheterotopic heart transplantation from Brown-Norway donors and receivedvarying, daily dosages of human recombinant G-CSF for 14 dayspostoperatively. No other treatment was administered except for G-CSF.Graft survival (days till cessation of graft heart beat), calculatedusing Breslow survival analysis, and AGC at six days post-operation weredetermined. Results are shown below in Table II.

                  TABLE II                                                        ______________________________________                                        Results  N Graft Survival                                                                           Survival Avg.                                                                             AGC × 10.sup.3 *                      ______________________________________                                        No treatment                                                                           6 6,7,7,7,7,8                                                                              7.0 ± 0.6                                                                              3.8 ± 1.0                                G-CSF 20 6 6,6,6,8,11,13                                                                            8.3 ± 3.0                                                                              2.9 ± 2.2                                μg/kg/day                                                                  G-CSF 250                                                                              6 6,11,12,12,15,18                                                                         12.3 ± 4 7.2 ± 3.2                                μg/kg/day                                                                  G-CSF 500                                                                              6 6,6,10,13,13,12                                                                          10.0 ± 3.2                                                                             14.9 ± 2.2                               μg/kg/day                                                                  ______________________________________                                         *The difference between GCSF treatment and no treatment was determined        with p < 0.05 using the Student ttest for groups with unknown                 distributions.                                                           

Treatment with G-CSF at 250 μg/kg/day resulted in a significantimprovement in graft survival (an average of 12.3 days, compared to 7.0days without G-CSF treatment). The improved graft survival observed withthe G-CSF-treated rats corroborates the reduction in the incidence foracute rejection noted in our clinical study in human liver transplantpatients.

What is claimed is:
 1. A method for treating acute rejection in anon-granulocytopenic organ transplant patient comprising administeringto the patient an amount of Granulocyte Colony Stimulating Factor(G-CSF) protein effective to reduce acute rejection.
 2. A method fortreating a non-granulocytopenic organ transplant patient comprisingadministering to the patient an amount of Granulocyte Colony StimulatingFactor (G-CSF) protein effective to reduce acute rejection.
 3. Themethod of claims 1 or 2 wherein the amount of G-CSF protein administeredis from 5 to 50 μg/kg/day.